摘要
目的: 探讨长链非编码RNA PCGEM1(long-chain noncoding RNA PCGEM1,Lnc RNA PCGEM1)通过TGF β2/Smad2信号通路调控食管癌(esophageal cancer,EC)细胞侵袭和转移。 方法: 收集62例EC患者癌组织及正常的癌旁组织,qRT-PCR检测Lnc RNA PCGEM1在EC及相应的癌旁组织、不同细胞中的表达情况;构建Lnc RNA PCGEM1沉默细胞系,细胞分为Eca-109-siPCGEM1组、阴性对照Eca-109-siNC组,并以Eca-109作为空白对照组,并构建Eca-109-siPCGEM1细胞的miR-148a沉默细胞系,分为siPCGEM1+simiR-148a组及siPCGEM1+siNC组;平板克隆实验检测Eca-109细胞增殖能力;划痕实验检测Eca-109细胞迁移能力的影响;使用生物信息学网站StarBase预测PCGEM1可互补结合的微小RNA(microRNA,miRNA),再根据Targetscan网站预测相应miRNA可靶向结合的基因;免疫印迹(Western Blot)检测TGF β2/Smad2信号通路蛋白表达情况。 结果: qRT-PCR结果显示,EC组织和食管癌细胞系中 Lnc RNA PCGEM1的表达水平高于癌旁组织(P<0.05),与其他细胞株相比, Lnc RNA PCGEM1在Eca-109细胞中表达量最高(P<0.05);与空白对照组相比,Eca-109-siPCGEM1组细胞克隆数和迁移数量明显减少,miR-148a表达量明显增加(P<0.05),空白对照组与Eca-109-siNC组相比差异无统计学意义(P>0.05);与siPCGEM1+NC组相比,siPCGEM1+simiR-148a组细胞克隆数、迁移数量明显增多(P<0.05);StarBase网站预测结果显示,Lnc RNA PCGEM1可与miR-148a互补结合,再经Targetscan网站预测分析显示,miR-148a与TGFβ2存在靶向结合位点;Western blot检测结果显示Eca-109-siPCGEM1组中TGF β2及Smad 2的表达明显低于空白对照组与Eca-109-siNC组(P<0.05),空白对照组和Eca-109-siNC组中上述指标的表达量无统计学意义(P>0.05)。 结论: Lnc RNA PCGEM1在食管癌中高表达,高表达Lnc RNA PCGEM1可能通过下调miR-148a水平,强化TGF β2/Smad2信号通路,从而促进EC的进展。
Objective: To investigate the regulation of long-chain noncoding RNA PCGEM1(Lnc RNA PCGEM1)in the invasion and metastasis of esophageal cancer cells through the TGF β2/Smad2 signaling pathway. Methods: Cancer tissues and adjacent normal tissues of 62 EC patients were collected. QRT-pPCR was used to detect the expression of Lnc RNA PCGEM1 in EC and corresponding paracancer tissues and different cells. Lnc RNA PCGEM1 silenced cell lines were constructed, and the cells were divided into Eca-109-siPCGEM1 group, negative control Eca-109-siNC group, and Eca-109 as blank control group.MiR-148a silenced cell lines of Eca-109-siPCGEM1 cells were constructed, which were divided into siPCGEM1+ simiR-148a group and siPCGEM1+siNC group.Eca-109 cell proliferation was detected by plate cloning assay.The effect of Eca-109 cell migration was detected by scratch assay. The bioinformatics website StarBase was used to predict the complementary binding microRNA (miRNA) of PCGEM1, and the targeted binding genes of corresponding miRNA were predicted based on the Targetscan website. Western Blot analysis of TGF β2/Smad2 signaling pathway protein expression. Results: QRT-PCR results showed that the expression level of Lnc RNA PCGEM1 in EC tissues and esophageal cancer cell lines was higher than that in adjacent normal tissues (P<0.05). Compared with other cell lines, Lnc RNA PCGEM1 had the highest expression in Eca-109 cells (P<0.05). Compared with the blank control group, the number of cell clones and migration in the Eca-109-siPCGEM1 group was significantly reduced, and the expression of miR-148a was significantly increased (P<0.05)..There was no significant difference between the blank control group and Eca-109-siNC group (P>0.05).Compared with the siPCGEM1+siNC group, the number of cell clones, number of migration were significantly increased in the siPCGEM1+ simiR-148a group (P<0.05). The prediction results of StarBase website showed that Lnc RNA PCGEM1 could be complementary to miR-148a, and the prediction analysis of Targetscan
作者
刘勇
王斌
金建琴
陈思思
LIU Yong;WANG Bin;JIN Jianqin(Shiyan People's Hospital Affiliated to Hubei Medical College,Hubei Shiyan 442000,China)
出处
《河北医学》
CAS
2020年第1期58-63,共6页
Hebei Medicine
